Pyrrole-mediated protein crosslinking: Toward an understanding of the molecular mechanism

The toxic metabolite of hexane, 2,5-hexanedione, and a group of related diketones lead to covalent crosslinking of proteins. The toxicity of a series of diketones shoes a strong correlation with the potency in crosslinking; however, the molecular basis of this crosslinking has not been fully characterized. The initial step involves the cyclization of the diketone with the amino group of lysyl residues. In a subsequent oxygen-dependent step, the protein-bound pyrroles lead to the covalent crosslinking. To study this second step, 6-(2,5-dimethylpyrrol-1-yl)norleucine (DMPN) was synthesized from [3H]-lysine, and the covalent binding of DMPN to ovalbumin was studied. DMPN bound covalently to protein over the same time period that protein crosslinking occurs. Since oxidized pyrrole species are electrophilic, sulfhydryl and amino groups are the most likely recipient protein nucleophiles of pyrrole binding. By competition assays, we found sulfhydryls to be more efficient than amines in competing with protein nucleophiles for the oxidized pyrrole. The results establish that both amines and sulfhydryls may serve as the nucleophile in pyrrole-mediated crosslinking. Although the sulfhydryl is the more efficient nucleophile in vitro, the process in vivo is likely to be dependent upon protein microenvironment. These findings are in conflict with the previous suggestion that crosslinking of pyrrolated proteins requires pyrrole-pyrrole interaction